A promoter is a sequence of DNA which can affect or control the level of transcription and which is responsible for (or provides the site for) the binding of RNA polymerase. The position of a promoter is fixed relative to the transcription start site within the genome of an organism. RNA polymerase is an enzyme (or a class of enzymes) which can bind to a promoter and bring about transcription of the structural gene (coding region) that is under control of the promoter, resulting in the production of messenger RNA (mRNA). Messenger RNA in turn provides the template for synthesis of polypeptides (translation).
Promoters have been studied in a variety of organisms, including viruses (e.g., U.S. Pat. Nos. 4,495,280, 4,518,690); bacteria (e.g., U.S. 4,551,433); plants; and animals. For a given species or type of organism, conserved regions of DNA (consensus sequences) have been found within promoters associated with a variety of structural genes. These regions are believed to be involved in the role played by the promoter in the transcription process.
Initiation of the transcription process in plants involves the interaction of a promoter with RNA polymerase II. Consensus sequences within plant promoters have been found at positions upstream from the 5' end of the transcription start point. There is a sequence of about seven base pairs positioned approximately 19-27 base pairs upstream of the transcription start point (i.e., positions -19 to -27) which is known as the TATA sequence, believed to play a role in RNA polymerase entry. There is another sequence of about nine base pairs positioned approximately 70 to 80 base pairs upstream from the transcription start point which is known as the CAAT box, believed to be involved in the regulation of the level of transcription. Other regions upstream of the transcription start point have been identified which affect the frequency of initiation of transcription in eukaryotes. These DNA sequences, known as enhancers or viral enhancer elements, have been found to affect the activity of promoters in their vicinity; these sequences are not promoters, as defined herein, in that their position need not be fixed. See H. Weiher et al., Science, 219, 626-631 (1983).
There have been studies of the introduction into plants of bacterial genes fused to bacterial promoters, resulting in expression of the bacterial gene in the plant. These introductions have involved the insertion of foreign DNA into the Ti plasmid of Agrobacterium tumefaciens, and the introduction of the foreign DNA into plants using Agrobacterium containing the modified Ti plasmid. See, e.g., R. T. Fraley et al., Proc. Natl. Acad. Sci., 80, 4803-4807 (1983).
In order to provide high levels of expression of foreign genes in plant cells it is desirable to isolate the promoter regions from strongly expressed plant genes and use these fused with the foreign gene coding sequence to direct high levels of expression. Certain polypeptides known to be highly expressed in plants have been the subject of considerable study. One of these is the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase). RuBPCase is the primary enzyme of the carbon fixation pathway in chloroplasts of plants of the C3 class. The enzyme consists of two types of polypeptide subunits, the small subunit (SSU) and the large subunit (LSU), eight of each of which assemble to give one molecule of RuBPCase. The small subunit, molecular weight approximately 14,000, is nuclear encoded and synthesized in the cytoplasm as a higher molecular weight precursor which includes a portion called the transit peptide. The precursor is transported into the chloroplasts via the mediation of the transit peptide. The precursor is processed to the mature subunit by post-translational mechanisms. The large subunit, molecular weight approximately 55,000, is encoded by chloroplast DNA and synthesized inside the chloroplast. The small subunit and large subunit are assembled in the chloroplast to yield RuBPCase.
RuBPCase is known to accumulate in response to light and studies have shown that there is a corresponding increase in the steady state levels of SSU mRNA resulting from increased transcription of the SSU gene. S. M. Smith et al., J. Mol. Appl. Genet., 1, 127-137 (1981) Studies have also shown that there are multiple copies of the SSU gene in the nuclear DNA of various plant genomes, including petunia (P. Dunsmuir et al., Nucleic Acids Res., 11, 4177-4183, 1983); pea (A. Cashmore et al., Genetic Engineering of Plants, T. Kosuge ed., 29-38, 1983); wheat (S. L. Berry-Lowe et al., J. Mol. Appl. Genet., 1, 483-498, 1982); and Lemna (C. F. Wimpee et al., Plant Molecular Biology, R. B. Goldberg ed., 12, 391-401, 1983).
In early work on the isolation of a cloned cDNA for the SSU gene in pea, there was a report of the sequence of a clone p20 corresponding to 123 amino acids of mature SSU, 13 amino acids of transit peptide, and 260 nucleotides of 3' non-coding region. J. Bedbrook et al., Nature, 287, 692-697 (1980). Subsequently, there were reports of sequence information for another pea cDNA clone, pSS15, and the corresponding genomic fragment pPS-2.4. G. Coruzzi et al., J. Biol. Chem., 258, 1399-1402 (1983); G. Coruzzi et al., EMBO J.,3 1671-1679 (1984). The isolation and characterization of petunia cDNA clones pSSU41, pSSU51, pSSU71, and pSSU117 and sequence information corresponding to part of the mature peptide region plus the 3' untranslated region for cDNA clones pSSU51 and pSSU117, was reported in P. Dunsmuir et al., Nucleic Acids Res., 11 4177-4183 (1983).
There have been reports of reintroductions into tobacco cells of promoter regions derived from pea SSU genes fused to bacterial gene coding regions. The introduction into tobacco, and expression, of a chimaeric gene consisting of the 5' region (promoter) of pea SSU gene labelled SS3.6 plus the coding region of the bacterial chloramphenicol acetyltransferase (CAT) and the 3' region of nopaline synthetase (nos) was reported in L. Herrera-Estrella et al., Nature, 310, 115-120 (1984). In this report the 5' region was fused to the coding region at a position 4 nucleotides upstream from the transcription initiation site. Another report disclosed the fusion of promoter and transit peptide DNA (plus the first codon--methionine--of the mature peptide region) from pea SSU gene SS3.6 to the structural gene for the bacterial protein neomycin phosphotransferase II, the introduction of the fused gene into tobacco plants, and the transport of the structural gene to the chloroplast in transformed plants. G. van den Broeck et al., Nature, 333, 358-363 (1985). A similar disclosure appears in P. H. Schreier et al., EMBO J., 4, 25-32 (1985), except that the promoter-transit peptide component contained additional DNA including the first 22 codons of the SSU mature peptide. There has also been a report of the introduction of DNA for pea SSU gene labelled pS4.0 into petunia, under the control of its own promoter, to yield heterologous RuBPCase containing pea SSU and petunia LSU. R. Broglie et al., Science, 224, 838-843 (1984).